We have identified a new class of molecular receptors amenable to drug targeting to modulate gene expression. Modulation of gene expression has broad applications in molecular therapeutics, including cancer, heart disease, inflammation, neurodegeneration, endocrinology, asthma, and diabetes. The scientific community affected by this discovery is very broad and includes basic as well as translational scientists. These molecular targets comprise secondary DNA structures (G- quadruplexes and i-motifs) found frequently in the promoter elements of a wide variety of genes including c-Myc, Bcl-2, VEGF, Hif-1a, PDGF-A, and RET. Proof of principle exists that stabilization of these secondary DNA structures with small molecules results in repression of gene expression in cells and this repression is dependent on an intact G-quadruplex in the promoter region. Quarfloxin, a drug originating from the PI's laboratory, is a first-in-class G-quadruplex-interactive drug that targets G-quadruplexes in ribosomal DNA and is in phase II clinical trials. The selectivity and corresponding lack of any serious toxicity in the clinical trials is partially dependent on the selective uptake into the nucleolus of cancer cells along with the 400-fold selectivity of Quarfloxin for the parallel-type G-quadruplex found in rDNA over duplex DNA. The objective of this proposal is to provide structural insight into silencer elements consisting of a G- quadruplex and i-motif within a duplex region that corresponds to the silencer elements. Herein lies the challenge because the i-motif is only normally generated under the negative superhelicity found in a plasmid supercoiled state or as a consequence of transcriptional runoff. We have proposed a solution to this problem along with techniques to provide a structure-based approach and development of a high-throughput assay for screening for compounds that interact with this composite structure. The ability to form the composite i-motif/G-quadruplex structure within a duplex molecule will permit us to identify small molecules that have enhanced target selectivity. The challenge addressed in this proposal is to design a system in which we can externally control the production of genes in cells. This technology, if successful, will have applications in the treatment of cancer, heart disease, and Alzheimer's disease.